1. Field of the Invention
The present invention concerns methods and means for controlling excessive proliferation and/or migration of smooth muscle cells, and in particular for treating stenosis, by using antagonists of a native ErbB4 receptor. The invention further concerns a method for the identification of ErbB4 agonists and antagonists capable of inhibiting or enhancing the proliferation or migration of smooth muscle cells.
2. Description of the Related Art
1. ErbB Receptor Tyrosine Kinases
Transduction of signals that regulate cell growth and differentiation is regulated in part by phosphorylation of various cellular proteins. Protein tyrosine kinases are enzymes that catalyze this process. Receptor protein tyrosine kinases are believed to direct cellular growth via ligand-stimulated tyrosine phosphorylation of intracellular substrates.
HER4/Erb4 is a receptor protein tyrosine kinase belonging to the ErbB family. Increased ErbB4 expression closely correlates with certain carcinomas of epithelial origin, including breast adenocarcinomas (Plowman et al., Proc. Natl. Acad. Sci. USA 90:1746-1750 [1993]; Plowman et al., Nature 366:473-475 [1993]). Diagnostic methods for detection of human neoplastic conditions (especially breast cancers) which evaluate ErbB4 expression are described in EP Pat Appln No. 599,274.
Other members of the ErbB family of receptor tyrosine kinases include: epidermal growth factor receptor (EGFR), ErbB2 (HER2/neu), and ErbB3 (HER3). The erbB1 gene encodes the 170 kDa epidermal growth factor receptor (EGFR) that has been causally implicated in human malignancy. In particular, increased expression of this gene has been observed in more aggressive carcinomas of the breast, bladder, lung and stomach (Modjitahedi, H. and Dean, C. (1994) Int. J. Oncol. 4:277-296). HER4 acts, in the absence of HER2, as a mediator of antiproliferative and differentiative response in human breast cancer cell lines. (Sartor et al., Mol. Cell Biol. 21:4265-75 (2001)).
The neu gene (also called erbB2 and HER2) encodes a 185 kDa receptor protein tyrosine kinase that was originally identified as the product of the transforming gene from neuroblastomas of chemically treated rats. Amplification and/or overexpression of the human HER2 gene correlates with a poor prognosis in breast and ovarian cancers (Slamon, D. J. et al., Science 235:177-182 (1987); Slamon et al., Science 244:707-712 (1989); and U.S. Pat. No. 4,968,603). Overexpression of HER2 (frequently but not uniformly due to gene amplification) has also been observed in other carcinomas including carcinomas of the stomach, endometrium, salivary gland, lung, kidney, colon, thyroid, pancreas and bladder.
A further related gene, called erbB3 or HER3, has been described. See U.S. Pat. No. 5,183,884; Kraus et al., Proc. Natl. Acad. Sci. USA 86:9193-9197 (1989); EP Pat Appln No 444,961 A1; and Kraus et al., Proc. Natl. Acad. Sci. USA 90:2900-2904 (1993). Kraus et al. (1989) discovered that markedly elevated levels of erbB3 mRNA were present in certain human mammary tumor cell lines indicating that erbB3, like erbB1 and erbB2, may play a role in human malignancies. They also showed that EGF-dependent activation of the ErbB3 catalytic domain of a chimeric EGFR/ErbB3 receptor resulted in a proliferative response in transfected NIH-3T3 cells. Furthermore, these researchers demonstrated that some human mammary tumor cell lines display a significant elevation of steady-state ErbB3 tyrosine phosphorylation further indicating that this receptor may play a role in human malignancies. The role of erbB3 in cancer has been explored by others. It has been found to be overexpressed in breast (Lemoine et al., Br. J. Cancer 66:1116-1121 (1992)), gastrointestinal (Poller et al, J. Pathol. 168:275-280 (1992), Rajkumer et al., J. Pathol. 170:271-278 (1993), and Sanidas et al., Int. J. Cancer 54:935-940 (1993)), and pancreatic cancers (Lemoine et al., J. Pathol. 168:269-273 (1992), and Friess et al., Clinical Cancer Research 1:1413-1420 (1995)). ErbB3 is unique among the ErbB receptor family in that it possesses little or no intrinsic tyrosine kinase activity (Guy at al., Proc. Natl. Acad. Sci. USA 91:8132-8136 (1994) and Kim at al. J. Biol. Chem. 269:24747-55 (1994)).
The ErbB receptors are generally found in various combinations in cells and heterodimerization is thought to increase the diversity of cellular responses to a variety of ErbB ligands (Earp et al Breast Cancer Research and Treatment 35: 115-132 (1995)). EGFR is bound by six different ligands; epidermal growth factor (EGF), transforming growth factor alpha (TGF−), amphiregulin, heparin binding epidermal growth factor (HB-EGF), β-cellulin and epiregulin (Groenen at al. Growth Factors 11:235-257 (1994)). A family of heregulin proteins resulting from alternative splicing of a single gene are ligands for ErbB3 and ErbB4. The heregulin family includes α, β and γ heregulins (Holmes at al, Science, 256:1205-1210 (1992); U.S. Pat. No. 5,641,869; and Schaefer et al. Oncogene 15:1385-1394 (1997)); neu differentiation factors (NDFs), glial growth factors (GGFs); acetylcholine receptor inducing activity (ARIA); and sensory and motor neuron derived factor (SMOF). For a review, see Groenen et al. Growth Factors 11:235-257 (1994); Lemke, G. Molec. & Cell. Neurosci. 7:247-262 (1996) and Lee at al. Pharm. Rev. 47:51-85 (1995). Recently three additional ErbB ligands were identified; neuregulin-2 (NRG-2) which is reported to bind either ErbB3 or ErbB4 (Chang et al Nature 387 509-512 (1997); and Carraway et al Nature 387:512-516 (1997)); neuregulin-3 which binds ErbB4 (Zhang at al. PNAS (USA) 94(18):9562-7 (1997)); and neuregulin-4 which binds ErbB4 (Harari at al Oncogene 18:2681-89 (1999)). HB-EGF, β-cellulin and epiregulin also bind to ErbB4.
While EGF and TGF do not bind ErbB2, EGF stimulates EGFR and ErbB2 to form a heterodimer, which activates EGFR and results in transphosphorylation of ErbB2 in the heterodimer. Dimerization and/or transphosphorylation appear to activate the ErbB2 tyrosine kinase. See Earp et al., supra. Likewise, when ErbB3 is co-expressed with ErbB2, an active signaling complex is formed and antibodies directed against ErbB2 are capable of disrupting this complex (Sliwkowski et al, J. Biol. Chem., 269(20):14661-14665 (1994)). Additionally, the affinity of ErbB3 for heregulin (HRG) is increased to a higher affinity state when co-expressed with ErbB2. See also, Levi et al., Journal of Neuroscience 15: 1329-1340 (1995); Morrissey et al., Proc. Natl. Acad. Sci. USA 92: 1431-1435 (1995); and Lewis et al., Cancer Res., 56:1457-1465 (1996) with respect to the ErbB2-ErbB3 protein complex. ErbB4, like ErbB3, forms an active signaling complex with ErbB2 (Carraway and Cantley, Cell 78:5-8 (1994)).
Because of the physiological importance, members of the ErbB family of receptor tyrosine kinases have often been targeted for therapeutic development. For example, Hudziak et al., Mol. Call. Biol. 9(3):1165-1172 (1989) describe the generation of a panel of anti-ErbB2 antibodies one of which, called 4D5, inhibited cellular proliferation by 56%. A recombinant humanized version of the murine anti-ErbB2 antibody 4D5 (huMAb4D5-8, rhuMAb HER2 or HERCEPTIN®; U.S. Pat. No. 5,821,337) is clinically active in patients with ErbB2-overexpressing metastatic breast cancers that have received extensive prior anti-cancer therapy (Baselga et al., J. Clin. Oncol. 14:737-744 (1996)). HERCEPTIN® received marketing approval from the Food and Drug Administration Sep. 25, 1998 for the treatment of patients with metastatic breast cancer whose tumors overexpress the ErbB21HER2 protein. Since HER2 is also overexpressed in other cancers, in addition to breast cancer, HERCEPTIN® holds a great potential in the treatment of such other cancers as well.
2. Smooth Muscle Cell Proliferation
Smooth muscle cells are very important structural and functional components of many hollow passages in the body, including blood vessels, gastrointestinal tract, airway passage (trachea and bronchi in lungs), urinary tract system (bladder and ureters) etc. They are responsible for elasticity that is so crucially required for normal functioning of these organs. They respond to a variety of physiological stimuli by constriction or dilation as needed, for example, for regulating the flow of fluids carried by them. They respond not only to chemical stimuli, such as growth factors and cytokines, but also to physical stimuli, such as pressure and stretch. Excessive proliferation of smooth muscle cells results in thickening of the wall and narrowing the lumen of the organs known as “stenosis” in a variety of disorders.
A number of growth factors and cytokines are implicated in the proliferation of smooth muscle cells. One category of such important molecules are EGF related ligands. For example, smooth muscle cells from a variety of such organs have been demonstrated to possess EGF receptors, and some of them even synthesize and secrete EGF ligands such as HB-EGF, thus setting up autocrine loop. Various EGF ligands act as potent mitogens and stimulate proliferation of smooth muscle cells often resulting in thickening of the wall and ultimately stenosis. For example, excessive proliferation of vascular smooth muscle cells (VSMC) is involved in pathology of vascular stenosis, restenosis resulting from angioplasy or surgery or stent implants, atherosclerosis, transplant atherosclerosis and hypertension (reviewed in Casterella and Teirstein, Cardiol. Rev. 7: 219-231 [1999]; Andres, Int. J. Mol. Med. 2: 81-89 [1998]; and Rosanio at al, Thromb. Haemost. 82 [suppl 1]: 164-170 [1999]). The thickening of blood vessels increases resistance to blood flow and ultimately leads to hypertension. Moreover, decreased blood supply to the tissue may also cause necrosis and induce inflammatory response leading to severe damage. For example, myocardial infarction occurs as a result of lack of oxygen and local death of heart muscle tissues.
Infantile hypertrophic pyloric stenosis (IHPS), which causes functional obstruction of the pyloric canal also involves hypertrophy and hyperplasia of the pyloric smooth muscle cells (Due and Puri, Pediatr. Res. 45: 853-857 [1999]). Furthermore, EGF, EGF receptor and HB-EGF are implicated in pathogenesis of pyloric stenosis (Shima et al., Pediatr. Res. 47: 201-207 [2000]).
Similarly, the urinary bladder wall thickening that occurs in response to obstructive syndromes affecting the lower urinary tract involves proliferation of urinary bladder smooth muscle cells. A membrane-bound precursor form of HB-EGF is expressed in urinary bladder smooth muscle cells and HB-EGF is a potent mitogen for bladder SMC proliferation (Freeman et al., J. Clin. Invest. 99: 1028-1036 [1997]; Kaefer et al., J. Urol. 163: 580-584 [2000]; Borer et al., Lab Invest. 79: 1335-1345 [1999]).
The obstructive airway diseases are yet another group of diseases with underlying pathology involving smooth muscle cell proliferation. One example of this group is asthma which manifests in airway inflammation and bronchoconstriction. EGF is implicated in the pathological proliferation of airway SMCs in obstructive airway diseases (Cerutis et al., Am. J. Physiol. 273: L10-15 [1997]; Cohen et al, Am. J. Respir. Cell. Mol. Biol. 16: 85-90 [1997]).
The instant invention discloses the use of ErbB4 receptor antagonists for controlling excessive migration and/or proliferation or smooth muscle cells and, in particular, for the treatment of stenosis.